This invention relates to methods for removing excess soluble protein from cellular lysates of Streptococcus pneumoniae (S. pneumoniae) serotypes used in the production of pneumococcal polysaccharides.
The capsular polysaccharide for each S. pneumoniae serotype utilized for vaccine products is produced by growing the organism in a complex liquid medium. The population of the organism is often scaled up from a seed vial to seed bottles and passaged through one or more seed fermentors of increasing volume until production scale fermentation volumes are reached. The end of the growth cycle can be determined by one of several means, at which point the cells are lysed through the addition of a detergent which aids in the cell wall breakdown and release of autolysin which causes cellular lysis when the cells reach stationary phase. The lysate broth is then harvested for downstream (purification) processing. This purification includes several column chromatography and diafiltration steps to recover the capsular polysaccharide that surrounds the bacterial cells. The polysaccharide, when conjugated with a high molecular weight protein, such as CRM197, and formulated into a vaccine containing conjugates of multiple serotypes, helps confer immunity (to S. pneumoniae) when injected into the target population, such as, for example, infants and young children.
Specifications have been set for the protein content in the purified polysaccharide of each serotype to reduce the risk of adverse events from the vaccine. For instance, in the currently marketed 7-valent pneumococcal conjugate (7vPnC) vaccine (Prevnar®), the specification for protein content in the purified serotype 4 polysaccharide is not more than 3%, and for the purified serotype 6B polysaccharide it is not more than 2% on a dry weight basis.
In some instances, it has proven difficult to remove the residual protein that is still present after the entire purification process. Efforts made to address this issue through changes in the purification processing of the cell lysate met with only moderate success.
It was therefore decided to attack this issue at the upstream side of the process. The key contaminant proteins were determined to be critical for cellular growth and integrity. Therefore, the remaining options available to reduce the total protein consisted of altering growth and/or harvest conditions.
The fermentation process is fairly straightforward. The cells (seed) are expanded in bottles of soy-based media, then passed through one or two seed fermentors, and finally passaged to a production scale fermentor. At each step the temperature and pH are closely monitored with pH being controlled by the addition of a base material (20% sodium carbonate). When the growth reaches a certain point, the run is ended by the introduction of a detergent, such as deoxycholate (DOC) sodium, which initiates a cell lysis process. After a hold period, the pH of the lysate broth is adjusted to 6.6 to precipitate the deoxycholate and cell membrane complexes. This material is held until processing by centrifugation and filtration can be carried out to remove the solids.
Much of the protein, however, remains solubilized in the clarified lysate, causing the residual protein content in the purified polysaccharide to exceed specification. Thus, there is a need to reduce the soluble protein levels in several pneumococcal serotypes during either the fermentation or purification process.